Molecular analysis and characterization of a protein involved in the replication of intracellular Toxoplasma gondii.
Previous studies in our laboratory have identified a cytoplasmic protein (p97) of T. gondii that is involved in the process of intracellular parasite replication. Monoclonal antibody inhibits parasite replication in vitro and recognizes a protein of approximate 97 kDa by Western blot analysis. Using biotinylation, we demonstrate that p97 is not expressed on the surface of the tachyzoite. Polyclonal sera raised against the purified native protein was used to isolate a cDNA of 3.3 kb from a library. The product of this gene expresses a protein of approximate Mr 97 kDa that is reactive to the antibody (1B8) raised against the native antigen. The protein sequence of this product suggests that it is within the cytoplasm as suggested by the lack of a signal sequence or hydrophobic trans-membrane domain. This protein fails to dissociate into a monomer in the presence of non-ionic detergents as shown by gel filtration and density gradient. Southern blot analysis demonstrates a homologous gene sequence in two closely related Apicomplexa, Neospora caninum and Besnoitia jellisoni suggesting this protein is conserved among certain species of the Sarcocystidae.
Matsuura
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Department of Medicine (Neurology), Dartmouth Medical School, Hanover, NH 03755, USA. tadashi.matsuura@dartmouth.edu
Kasper
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000913:Antibodies, Protozoan / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology; D016782:Apicomplexa / Q000235:genetics; D001483:Base Sequence; D019921:Biotinylation; D015139:Blotting, Southern; D003593:Cytoplasm / Q000737:chemistry; D016054:DNA, Protozoan / Q000235:genetics; D006790:Host-Parasite Interactions; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D017422:Sequence Analysis, DNA; D012995:Solubility; D014122:Toxoplasma / Q000737:chemistry / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D015800:Protozoan Proteins